WO2014166535A1

WO2014166535A1 - An apparatus for detection, identification of molecules and sequencing of dna, rna or other natural or artificial polymers using graphene and a laser light beam.

Info

Publication number: WO2014166535A1
Application number: PCT/EP2013/057520
Authority: WO
Inventors: Heiko Schwertner
Original assignee: Heiko Schwertner
Priority date: 2013-04-10
Filing date: 2013-04-10
Publication date: 2014-10-16

Abstract

A sample containing single molecules (4) such s DNA is placed on a graphene layer (1). The surface is scanned with a laser beam (6). This light beam induces in the graphene layer a stream of electrons, which are transported in two dimensions. By measuring the voltage or current, the system is able to determine the origin of the laser beam contact point (9) on the graphene layer (1). In cases the beam hits a molecule or a monomer of the polymer, the voltage or current signal changes. By means thereof the system is able to determine the location of the target molecule (4) on the graphene layer (1). Moreover, a light emission signal is detected (5).

Description

l

Description

An apparatus for detection, identification of molecules and sequencing of DNA, RNA or other natural or artificial polymers using graphene and a laser light beam.

Technical Field

[0001] The technical field is the detection, identification of small molecules and determination of the chronology of monomers in DNA or RNA both natural polymers and other natural or artificial polymers.

[0002] Detection and identification of small molecules in mixtures is the basic knowledge in all fields of chemical and pharmaceutical analysis. Several different methods exist for different kinds of usage and environmental conditions. It is an important field in science.

[0003] Sequencing (determination of the chronology of monomers in a polymer) technology of polymers is an important knowledge field of the chemical and physical properties of natural and artificial polymers. Artificial polymers are all the different kinds of plastics like PP, PE, PET, PS .etc. Natural polymers are DNA, RNA, peptides, proteins, poly carbohydrates etc.

[0004] In particular DNA and RNA is a field of fast and deep innovation. Target of these investigations is to show a new fast and cheap method of sequence determination (short: sequencing) of DNA and/or RNA and other natural or artificial polymers.

[0005] The sequence information is used for a better understanding of

biochemical pathways, to find new illness treatments, for identification uses, personalized health care and more.

Background Art

[0006] During the past century several methods to determine the sequence of natural and artificial polymers have been developed. In particular for sequencing of DNA methods following methods have been developed:

• Maxam-Gilbert sequencing

Chain-termination methods

• Dye-terminator sequencing ^• Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS) Polony sequencing

• 454 pyrosequencing

llumina (Solexa) sequencing

• SOLiD sequencing

Ion semiconductor sequencing

DNA nanoball sequencing

• Helioscope(TM) single molecule sequencing

• Single Molecule SMRT(TM) sequencing

Single Molecule real time (RNAP) sequencing

• Nanopore DNA sequencing ZHOU J., et al. Recent patents of

nanopore DNA sequencing technology: progress and challenges. Recent Pat DNA Gene Seq.. Nov 2010, vol.4, no.3, p.192-201.

• Sequencing by laser scanning

[0007] All these methods have been briefly described in an article on Wikipedia SEVERAL AUTHORS, et al. Internet Link:

http://en.wikipedia.org/wiki/DNA_sequencing. Wikipedia. 2013. In the literature listed in this online article, the methods have been explained in detail. All these methods are State of the Art, but none of these methods uses graphene with exemption of the Nanopore DNA sequencing DANIEL BRANTON, et al. The potential and challenges of nanopore sequencing. Nature Biotechnology. 2008, vol.26, p.1146 - 1153.

[0008] All these methods have disadvantages like short reading length, need for expensive and technical complicate equipment, destroying of the DNA / RNA samples or high failure rates. The most important disadvantage is that they can only sequence, but they cannot identify or detect other molecules than the source material like DNA or RNA.

[0009] For detection and identifying small molecules, a wide range of methods has been published and used over the past hundred years. In the last years, methods using graphene have been published. SCHEDIN, F., et al. Detection of individual gas molecules adsorbed on graphene. Nature Materials. 2007, vol.6, p.652-655 and OKAMOTO SHOGO, et al. Fragment-Modified Graphene FET for Highly Sensitive Detection of Antigen-Antibody Reaction. IMCS. 2012, vol.DOI 10.516, no.6.1.3, p.519- 522.

[0010] MAO. S., et al. Specific protein detection using thermally reduced

graphene oxide sheet decorated with gold nanoparticle-antibody conjugates.. Adv. Mater.. 2010, vol.22, no.(32), p.3521-3526.

[0011] DAN DU, et al. Sensitive Immunosensor for Cancer Biomarker Based on Dual Signal Amplification Strategy of Graphene Sheets and Multienzyme Functionalized Carbon Nanospheres. Anal. Chem. 2010, vol.82, p.2989- 2995.

[0012] All these methods modify graphene or use direct voltage current change.

No method uses unmodified graphene with a laser beam for the detection and identification of molecules.

[0013] In the recent past, the chemical substance graphene has been target of a number of scientific investigations because of the easy availability of this material and the physical and chemical properties of graphene. An overview about the properties of graphene has been briefly described in an article on Wikipedia. SEVERVAL AUTHORS, et al. Internet Link:

http://en.wikipedia.org/wiki/Graphene. Wikepedia. 2013. In the literature listed in this online article, the methods have been explained in detail.

[0014] Two properties of graphene are important in relation with identification and detection of small molecules or sequencing of natural (DNA / RNA, proteins, sugar chains etc.) or artificial polymers,

[0015] Firstly, graphene can convert light into electricity. This property allows inducing a voltage change into a layer of grapheme.

[0016] BONACCORSO, F., et al. Graphene photonics and optoelectronics, nature photonics. 31 Aug 2010, vol.NPHOTON.20, no.DOI: 10.10, p.611-622 and literature cited in. This property is today used for photovoltaic solar cells, photo detectors and touch screens.

Secondly, graphene transports electrons only in two dimensions in a small band. ULBRICHT, GERHARD. 2-dimensionaler Ladungstragertransport. Dissertation of the Max-Planck-lnstitut fur Festkoperforschung (Stuttgart (DE)). 29.Sep.2008, p.1-270. This property allows detecting electrons inducted by laser light and to determine the origin of the point where laser light crosses the graphene layer.

[0017] Graphene is used for DNA sequencing using a method called nanopore sequencing. DNA can pass through the nanopore for various reasons. For example, electrophoresis might attract the DNA towards the nanopore, and it might eventually pass through it. Or, enzymes attached to the nanopore might guide DNA towards the nanopore. The scale of the nanopore means that the DNA may be forced through the hole as a long string, one base at a time, rather like thread through the eye of a needle. As it does so, each nucleotide on the DNA molecule may obstruct the nanopore to a different, characteristic degree. The amount of current which can pass through the nanopore at any given moment therefore varies depending on whether the nanopore is blocked by an A, a C, a G or a T. The change in the current through the nanopore as the DNA molecule passes through the nanopore represents a direct reading of the DNA sequence. Alternatively, a nanopore might be used to identify individual DNA bases as they pass through the nanopore in the correct order. The potential is that a single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification step or a chemical labeling step or the need for optical instrumentation to identify the chemical label. SEVERAL AUTHORS, et al. Internet Link:

http://en.wikipedia.org/wiki/Nanopore_sequencing. Wikipedia. 2013 and the literature listed in this online article),

[0018] Another method related to this invention is sequencing by scanning a

polymer US 6.524.829 B (STEFAN SEEGER ) 25.02.2003 Microscopy- based techniques, such as AFM (transmission electron microscopy) that are used to identify the positions of individual nucleotides within long DNA fragments (>5,000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording.

[0019] One method to sequencing DNA strands is by using a STM (scanning tunneling microscope) technique. For this method, ultrapure DNA is required. PORATH, DANNY, et al. Scanning tunnelling microscopy: A DNA sequence scanned. Nature Nanotechnology. 2009, vol.4, p.476-477.

[0020] No patent or literature describes the use of graphene scanned by a light beam to analyses the molecule laying on the graphene surface, but a few inventions are referencing between graphene and sequencing:

[0021] KR 102011036204 A (SEO, TAE SEOK) 01.10.2009

[0022] WO 002011123513 A (JOHNSON ALAN T.) 30.03.2011

[0023] CN 000101846648 A

[0024] CN 000101805432 A

[0025] CN 000101789440 A

[0026] US 020100028573 A (JAE-KAP, LEE ) 04.02.2010

[0027] WO 002012005857 A (PRESIDENT AND FELLOWS OF HARVARD

COLLEGE) 08.06.2010

Disclosure of Invention

[0028] The state of art methods used for sequencing have disadvantages like:

- Disruption of the polymer chain (e.g. DNA, RNA)

- Use of enzymes or reagents for sequencing

- High cost through the need of several handling steps and a complex instrument base

- Less safe and accurate because it is not possible to repeat the

sequence on the same probe

- Natural and artificial polymers cannot be sequenced with the same sequencing methods

- Need of a distinguished starting point or a guidance of the DNA strand

- No method can in parallel make a sequence determination and a

detection / identifying of molecules

- Impossibility to detect and identify small molecules in the same

analytical run

- A relatively high concentrated probe is needed and the probe has to be clean [0029] The need of nanopore sequencing is to guide the polymer to and trough the nanopore, so no enzymes or chemicals are necessary. In addition to the starting point, an auxiliary molecule is needed to guide the polymer through the nanopore and to initiate the sequencing process. But the measureable signal is very low. To increase the signal intensity multiple polymer molecules have to move simultaneously through the graphene pores. The disadvantage is that the signal to noise ratio degraded dramatically, because the nanopore sequence needs a starting point on a nucleotide base. The oxford nanopore technologies sequencing

technology solves this problem by using enzymes which captured and guided the DNA or RNA strand through the nanopore. In contrast to the nanopore sequencing with grapheme, the Oxford nano sequencing method uses a modified natural protein pore. The main disadvantage is that the pore protein and the guiding enzyme are not stable (shelf life less than 6 hours) and the variability of such natural raw materials is a problem. The single experiment cannot by repeated exactly in the same way. The result of these disadvantages is a higher failure rate (minimum 5%).

[0030] . For the industrialization of both methods, pore sequencing via graphene pore or via enzyme pore, a higher effort is needed than by using the sequencing method described in this invention.

[0031] The DNA scanning method with 3TM at this stage can only be

implemented with complex and expensive STM apparatuses, and it is also relatively slow with a very low read length. Consequently, as the authors indicate, considerable developments are necessary before this approach can be carried over into any practical application. Although it is unclear whether this method will ever be a leading sequence procedure, the direct imaging reported by Tanaka and Kawai is unprecedented for DNA— this in itself is a considerable scientific achievement. [0032] All the existing sequencing methods need pre-cleaned DNA / RNA probes in a high quality and to determine this grad of cleanness a separate method is needed. In addition to the cleanness of the probe a relatively high concentration of the target molecule is a must, otherwise the classical methods does not work.

[0033] Solution of the Problem

[0034] The method described in this invention solved the above listed technical problems by using graphene and laser light. Preferably a graphene mono layer is needed for the invention, but a multilayer of graphene can be used also. In the literature, several methods of production and handling of graphene are described incl. the mass production of graphene in different qualities and quantities.

[0035] A laser beam light can induce electrons measurable as an electric signal in the graphene layer (Fig.1). Newly a science group shows that one photon can activate several electrons TIELROOIJ, K.J., et al. Photoexcitation cascade and multiple hot-carrier generation in graphene. Nature Physics . 23 Jan 2013, vol.nphys2564, no.doi:10.103. This physical property allows to enhance the efficiency of the system and also to determine the 3D structure of the outer phase of a molecule.

[0036] The origin point of this electric signal (voltage or current) can be

determined by the measurement of the electric signal if two electrodes are connected to the graphene the principle is illustrate by a resistive touch screen principle (Fig.2).

[0037] The graphene layer used in the invention has on each side a conductive path (Fig.1) or a connection to conductive surfaces. The graphene layer has to be connected preferably to two separated conductive paths or paths pairs or a connection to two separated conducting surfaces or surface pairs. This part secures the detection of the electrons induced by the light beam hitting the graphene layer. [0038] The graphene layer is preferably a mono layer without disruptions or holes. The invention is able to use also multiply graphene layers incl. partial multiply graphene layers, preferably without disruptions or holes. The conductive paths can be also used to tread the graphene layer with an alternating voltage (AC voltage).

[0039] A probe containing small molecules and/or DNA, RNA or other polymers will be placed on the surface of the graphene layer; single strand DNA or RNA is preferred if sequencing is main usage (Fig.3).

[0040] The DNA, RNA or other polymers interact or bind to the graphene layer.

Pre cleaned DNA, RNA or other polymers are preferred, because they enhance the performance of the test and reduce the signal to noise ratio.

[0041] To reduce the possibility of engulfed polymer strands the graphene layers are treated with an alternating voltage during the application of the polymer containing probe or after the application of the probe.

[0042] DNA, RNA or other polymers which are to be sequenced are preferred in a single strand form, but the system used in this invention, can utilize double strand DNA, RNA or pre- or post-modified DNA or RNA also. Pre modifying of DNA or RNA and other polymer means that the probe applied on the graphene layer has been chemically or physically modified, for example by a covalent binding of a fluorescence marker molecule to the DNA or RNA strand or binding a complementary primer. Such chemical or physical modifications can be done after the application of the probe also - a post-modification.

[0043] After the application of the polymer containing probe a laser beam of the system scans the surface of the graphene layer in xy direction (Fig.3/9). Thereby the system is able to determine a measurable continuous electric signal. If the laser beam crosses the single strand DNA / RNA or polymer, the electric signal changes. This change can be measured and separated from the signal before. By a computer result interpretation the xy orientation of the DNA, RNA or other polymer strand can be determined. Preferably to enhance the signal to noise ratio the graphene layer is scanned before the molecule containing probe will be applied on the graphene layer. This step provides information about surface defects, already existing impurities and gives over the complete graphene surface zero values.

[0044] The system does not need a light beam with a diameter in the range of one nucleotide. The preference is to have a small diameter of a light beam, but in the scanning mode it is possible to make a mathematical differentiation to one nucleotide in a chain. In a case of step mode, an area of interest can be pointed on several parts of this area and the resulting spectra can be analyzed also by mathematical algorithms.

[0045] With this scanning method, all kinds of molecules that lay on the graphene layer can be located. When the laser beam crosses a molecule, the change of the electric signal is measured. It does not only provide information about 2 dimensions of the molecule, but also of a third dimension. The height of a molecule changes the electric signal in a mathematical function of the height of the molecule.

[0046] If the light beam crosses a single molecule or a polymer chain, light is

reflected or emitted like fluorescence emission. This light is detected by the detector and the resulting spectra are analyzed by mathematical algorithms. This procedure allows receiving more information than by the unique measurement of a single light wave, but it is also a possible method for analyzing. Analyzing a single wavelength gives an advantage in the speed and lower amount of the data which have to be calculated. Both analytical methods can be combined, but getting and analyzing the spectra is the preferred analyzing method.

[0047] In case the laser beam has a distinctive wavelength, preferably 267nm, or a range, preferably between 260 and 271 nm, the emission light

wavelength can be measured, if the beam crosses a monomer in the polymer that has a fluorescence properties like DNA or RNA RANDOLPH, J.B., et al. Stability, specifity and fluorescence brightness of multiply- labeled fluorescent DNA probes. Nucleic Acids Research. 1997, vol.25, no.14, p.2923-2929. Each nucleotide of DNA or RNA has a different unique emission spectrum WARD, D.C., Reich, E.. Fluorescece Studies of Nucleotides and Polynuceotides. The Journal of Biological Chemistry. 1969, vol.244, no.5, p.1228-1237. ONIDAS, D., et al. Fluorescence Properties of DNA Nucleosides And Nucleotides: A Refined Steady-State and Femtosecond Investigation. J. Phys. Chem. B. 2002, vol.106, p.11367-11374. It can identify directly from the light wave emission when the laser beam crosses the monomer.

[0048] Preferably one laser beam is used in the system, but two or more separate laser light beams can be used in the system as well. Preferably these laser beams are used one after another, but it is not necessary. It depends only on the number or kind of the light detectors linked in the system.

[0049] The fluorescence properties of a molecule can be activated by a laser beam which is autonomous from the scanning laser beam. The scanning laser beam gives the xy-coordi nates of a detected molecule and additional information of the z-coordination. With this information the independent second laser beam can be exactly focused on the target molecule to activate fluorescence properties or other light inducible properties of the molecule.

[0050] With the combination of the known location and the fluorescence emission signal at this point it is possible to determine the sequence of monomers in a polymer or to identify a single molecule. It is also possible to determine the chronology of short chains of molecules.

[0051] The laser orientation and the contact point on the graphene layer can be mathematically calculated from the hardware system data, by determining the adjustment of the instrument laser beam. This information can be used additionally to verify the measured contact point of the laser beam.

[0052] The probe, for example DNA or RNA will not be destroyed by using the method, therefore the scanning part and/or second part, the activating of fluorescence or other light inducible properties of the molecule, can be done several times again. This possibility allows increasing the signal to noise ratio and/or to verify the results. This is a possibility that no other actual method can provide. The probe itself can be used, after the experiment, for other methods like PCR or cleaving methods etc.

[0053] The preferred method is to scan the surface with one laser beam which has a distinctive wavelength. It is also possible and represents a part of this patent, to use two or more laser beams with different wavelengths (Fig.4).

[0054] If, according to the patent, two or more laser beams are used, each single beam can be used for different tasks. For example, one laser beam can be used for scanning the graphene and another can be used for inducing the fluorescence emitting light from the nucleotides of the DNA. To induce the fluorescence emitting light, more laser beams can be used for each different nucleotide. All these methods that differ from the preferred method with a single beam can be used to enhance the performance of the sequencing results.

[0055] In case a laser beam hits a molecule the intensity of the electric signal induced in the graphene layer declines dependent on the thickness of the molecule. The analysis of the thereby obtained data and information and in combination with the data resulting from the scans, the system is able to identify a molecule not only by the emission properties. Analyzing these data the 2D and 3D properties of the surface of a molecule can be determined.

[0056] If the amount of data is too high for an acceptable data analysis, the laser beam can be used to cut out the contour of the target molecule or smaller areas. After the cutting the target molecule lays on a considerably smaller space. This space can by analyzed, like described beforehand, but now the amount of data is dramatically smaller than before.

[0057] Advantageous Effects of Invention

[0058] The usages of graphene, described in this invention, provides several advantages:

- One advantage is that graphene stabilizes polymers incl. DNA or RNA as a result of its passive physiochemical properties.

- Graphene reduces scattered light by adsorbing. This effect enhances the efficiency of the fluorescence detector because in case the laser beam crosses the molecule all the light will be reflected or a fluorescence emission will be activated. The light of the laser beam which strikes before or after the molecule is adsorbed by the graphene. The result is an improvement of the signal to noise ratio. This is also an advantage of the passive physiochemical properties of graphene.

[0059] It is possible to calculate the exact location on which the laser beam

strikes the graphene layer. The point of impact can be measured by the described method. If both parts of information are combined, it is possible to confirm and verify the measurement.

[0060] The treatment of the graphene layer with the polymer - during or after the application - with alternating voltage (AC voltage) has the positive effect that the polymer strand will be stretched. This active stretching by AV voltage decreases the amount of engulfment and other negative

orientation of polymers like DNA or RNA.

[0061] The combination of graphene and laser scanning gives a lot of

advantages. The list below gives an overview:

• Except for the preparation steps and enzyme treatment, no chemical reaction is necessary

Method can be used for identification of small molecules too (see 13.)

• Sequencing of a polymer and simultaneously detection and identifying of smaller molecules is possible

• DNA /RNA will not be destroyed and can be used for further

manipulation steps

• The sequence determination can be done several times to enhance the rate of reading accuracy

Fast measurement is possible: Laser guided speed (more than 10.000 bp/sec)

• Extreme long read length possible (million bp)

• Extreme small volume necessary (less 1 pi) -> less probe volume

Complete integrated work flows possible

• Same method for DNA, RNA, carbohydrates, artificial polymers or natural polymers

• The Hardware is cost effective (graphene sensor, laser (LED), detector with no moving part and small • The workflow is cost effective (probe preparation / cleaning, starting point fixation, measurement, result interpretation)

• No cleavage of DNA or RNA is necessary but a single DNA/RNA

strand is preferred

No enzyme needed (more stability, easy transportation, no buffer change, less intra assay variance, etc.)

• Scanning a bigger region of the graphene layer, the composition of the probe can be determined.

Polymer capturing by modifying the graphene layer with antibodies,

DNA / RNA primers and natural or artificial molecules

Method of the invention does not need a starting point or a guidance of the polymer by enzymes or physical methods.

Multi scanning of the same allows to improve the detection quality and to verify the results.

[0062] Other 2-D material can be used in the way as graphene is used in this invention RAMAKRISHAN MATTE H.S.S., et al. MoS2 and WS2 Analoges of Graphene. Angewandte Chemie. 2010, vol.122, p.4153-4156. The uses of MoS2, WS2 or other 2-d materials are also a part of this invention.

These materials can replace the before describe graphene layer, where be the graphene is the preferred material. Carbon nanotubes lay on a surface, preferred in one layer and tube by tube, are also a part of this invention and can also replace the graphene layer.

Brief Description of Drawings

[0063] Fig.1 describes the induced electron activation and movement by a light beam in a graphene mono layer. The numbers in the following table gives a brief description of the drawing parts.

No. Description Conductive path 1

2 Conductive path 2

3 Graphene layer

4 First contact point / point of origin of the laser beam

5 Light source (preferred laser)

6 Laser light induced electron transport in the

graphene layer

7 Moving direction of the light induced electron

[0064] Fig .2 describes the principle of a resistive measurement in a two

dimension layer. It is an example how the measurements of the electronic signal are transcribed in a xy-orientation. The measurement itself is described in Fig.3 and Fig.4. The numbers in the following table give a brief description of the drawing parts.

Description

Conductive path

Resistive layer 1

Resistive layer 2

Origin/location of the primary signal

[0065] Fig.3 illustrates the graphene layer with DNA laying on the surface. The figure includes also the laser scanning unit, scanning the area with the DNA probe. The numbers in the following table give a brief description of the drawing parts.

No. Description

1 Conductive path 1 Conductive path 2 Graphene

Frist contact point / point of origin

Light source (preferred laser)

Through light induced electron in graphene layer

Moving direction of the light induced electron

Starting point for the movement

Moving directions

Light detector

Emitting fluorescence light

Fig.4 describes the complete system incl. the graphene layer, the scanning laser beam, the detection unit and the conductive paths, (see also Fig.3). The numbers in the following table give a brief description of the drawing parts, (see also Fig.2)

Description

Graphene layer

Conductive path

Single strand DNA

Photomultiplier detector

Light source (preferred laser)

Through light induced electron in graphene layer

Moving direction of the light induced electron

Point of origin

Example of a sequencing unit according to the invention

Principle of resistive measurement method for determination of the origin Best Mode for Carrying Out the Invention

[0067] The preferred method carried out in this invention uses graphene layer scanned by a laser beam. The graphene layer is in contact with two conductive paths. The system includes a light detector unit, which allows detecting reflecting or emission lights, while the laser beam scans the graphene surface.

[0068] If a measurement is carried out in case of the system described before, the surface of the graphene layer is scanned with a laser beam before the probe is applied. By doing this a zero value is determined. Afterwards the highly diluted target molecule solution is applied on the graphene layer. After removal of the solvent, the target molecules are bound to the graphene layer. Finally, the surface of the graphene layer is scanned a second time.

[0069] The location of a target molecule on the surface can be measured by the induced electrons in the graphene and by detection of the reflecting or emission light. On the basis of these data, physical properties of the target molecule can be determined.

[0070] By combining both results, the location on the one hand and the physical property on the other hand, a target molecule can be identified and/or sequenced.

[0071] The method can be used for identifying small molecules, determination of composition of a fluid and sequencing of a natural or artificial polymer.

Mode(s) for Carrying Out the Invention

Several modes for carrying out the invention were describe based on working instructions

Example 1 :

[0072] Analyzing DNA / RNA single strand with pre-cleaning [0073] DNA containing probes is pre-cleaned with established laboratory methods. The DNA containing buffer is replaced by a vacuum removable buffer.

[0074] The system makes a pre-scan of the graphene layer without a probe on the graphene layer. The received data are used later in the mathematically analyzing of the complete data set.

[0075] The cleaned DNA in buffer is placed on the graphene sequencing device.

After incubation by room temperature, DNA or RNA attach to the graphene layer. The fluid is removed by a slight vacuum and/or temperature.

[0076] A surface fast-scan secure is started. This fast-scan gives the following information:

- Cleanness of the surface

- Rough location of the target molecules

- Status of the linearity of the target molecule

- Amount of the practical usable areas

[0077] After the fast-scan, a detailed scan of the near area of the target molecule or molecules is following. This mayor scan gives following information:

- Sequence of a single molecule

- 2D and 3D structure of a single

- Length of a single molecule

[0078] The resulting data is mathematical analyzed. That can be done parallel or after the technical measurement. This calculation provides following information:

- Length of the polymer

- Sequence of the polymer

- Sequence variance of polymers

[0079] Washing steps can be additional made by each single step, to increase the quality of the workflow steps.

[0080] In case of unclear or bad measurement the total process can be repeated incl. a chemical treatment, because the polymer will not be destroyed using this method. If the process is well done, the polymer can be used in further experiments by detachment of the target molecule from the graphene layer. Example 2:

[0081] Analyzing DNA double strand and/or RNA without a pre-cleaning step [0082] According to example 1 the main workflow will be the same with the

exemption of step 2:

Table 1

# Workflow Step

1 DNA containing probes collected

2 The DNA containing probe is highly diluted in a vacuum removable buffer

3 Placing of the solution on the graphene sequencing device

4 Incubation by room temperature

5 Removing of the buffer by a slight vacuum and/or temperature

6 Pre-scan

7 Mayor scan

8 Mathematical analysis and interpretation of the results

[0083] In case of unclear or poor measurement the total process can be repeated incl. the chemical treatment, because the polymer is not destroyed by using this method. If the process is well done, the polymer can be used in further experiments after detachment from the graphene layer.

Example 3:

[0084] Analyzing of an artificial polymer to determine the orientation of the side chain functionality

[0085] According example 1 the main workflow is predominantly the same except step 1 & 2:

Table 2

Workflow Step

Artificial polymer is dissolved in a probate organic solution which is vacuum removable

2 The dissolved probe is highly diluted

3 Placing of the solution on the graphene sequencing device

4 Incubation

5 Removing of the organic solution by a slight vacuum and/or temperature

6 Pre-scan

7 Mayor scan

8 Mathematical analysis and interpretation of the results

[0086] In case of unclear or poor measurement the total process can be repeated incl. the chemical treatment, because the polymer is not be destroyed by using this method. If the process is well done, the polymer can be used in further experiments after detachment from the graphene layer.

Example 4:

[087] Analyzing of the primary sequence of Protein or Peptide

[088] According example 1 the main workflow is predominantly the same except step 2:

Table 3

# Workflow Step

1 Protein or Peptide containing probe collected

2 The Protein or Peptide containing probe is highly diluted in a vacuum removable buffer.

3 Placement of the solution on the graphene sequencing device.

4 Incubation by room temperature

5 Removing of the buffer by a slight vacuum and/or temperature.

6 Pre-scan

7 Mayor scan

8 Mathematical analysis and interpretation of the results [089] In case of unclear or poor measurement the total process can be repeated incl. the chemical treatment, because the polymer is not destroyed by using this method. If the process is well done, the polymer can be re-used in further experiments after detachment from the graphene layer.

Example 4:

[090] 3D scanning of a DNA, RNS or other polymer

[091] The system contains in minimum two laser beams with different

wavelengths.

[092] According to example 1 the main workflow is predominantly the same with the exemption of step 2:

Table 4

# Workflow Step

1 Protein or Peptide containing probe collected

3 Placement of the solution on the graphene sequencing device.

4 Incubation by room temperature

5 Removing of the buffer by a slight vacuum and/or temperature.

6 Pre-scan with laser 1.

7 Main scan with laser 1.

8 Second scan with laser 2 (this laser has an different angle to laser 1)

9 Comparison of the results

8 Mathematical analysis and interpretation of the results

[093] In case of unclear or poor measurement the total process can be repeated incl. the chemical treatment, because the polymer is not destroyed by using this method. If the process is well done, the polymer can be re-used in further experiments after detachment from the graphene layer. Industrial Applicability

[094] Especially DNA and RNA is a field of fast and intensive research. Target of the industrial investigators is to find a fast and cheap method of sequence determination of DNA and/or RNA. The sequence information is used for a better understanding of biochemical pathways, to find new treatments against cancer or other health deficiencies, for identification of virus or bacterial or other biological targets, in personalized health care to optimize the drug treatment.

[095] Knowledge of DNA sequences has become indispensable for basic

biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics. The advantages of DNA sequencing have significantly accelerated biological research and discovery. In particular the pharmaceutical (human and veterinarian) and the agriculture industry have a high interest in a fast, cheap, stable and easy to use method for sequencing of natural and artificial polymers. At the moment sequencing of DNA and RNA attracts the most attention, because of the possibilities it offers in the segment of personalized health care. The worldwide health care market volume is around 5.7 trillion US$ J.

KARTTE, et al. Studie "Weltweite Gesundheitswirtschaft -Changen fur Deutschland" im Auftrag des BMWi. BMWi - Literature Service. 17. Aug. 2011. With the knowledge of DNA and/or RNA sequences cost and efficiency of therapies can be overall decreased and in addition it gives the hope to find new possibilities of treatment against cancer. The agriculture industry is interested in this knowledge for the optimization of crops and for the overall improvement of animal health.

[096] Direct industrial applications are:

Identification of viruses, bacteria, fungi, cancer cells

• Identification of diseases

Forensic analysis

• Determination of age-related changes of the genome or the resulting proteome • Sequencing of DNA / RNA

• Determination of the level of purity of organic and inorganic mixtures

• Simultaneously detection / identification of molecules and

determination of DNA/RNA strands or artificial polymer sequences

• Usage in Point of care applications

• Analytical part of the personalized health care

Sequence Listing Free Text

[097] No sequence is attached to this invention

References

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Claims

1. An Apparatus for detection, identification, determination of 2D/3D surface

properties, location, orientation and determination of chemical and physical properties of single molecules and/or sequencing natural or artificial polymers using one or more graphene layers, a light beam scanning unit, which scans the surface point by point or in a continuous way and a light detector unit from a composition, comprising:

- One or more separated graphene layers, wherein the graphene layer contains one or more stacked graphene layers or functionalized graphene monolayers or multilayers of graphene with or without or within additional ions or molecules between them, where the graphene layer unit can be a fixed part of the instrument or an external part.

- A light beam source, wherein one or more laser beams preferable with different and distinctive wavelength are able to scan continuously or point by point the surface of the graphene layer.

- A detector for electrons, wherein the graphene layer or layers are in a minimum contact with one pair or two pairs of conductive paths or laying on an electron detector, which allows the measuring of an electric signal induced by the light beam source and/ or electron source.

- A detector for light, wherein the detector is able to analyze wavelength of reflecting and emission light.

- A computer for mathematical analysis of data from the electron and light detector and controlling and timing of the apparatus.

2. The apparatus defined in claim 1 wherein said use for identification and/or sequencing can be used to sequence unmodified, pre/ or post modified artificial and natural polymers like, dsDNA, ssDNA, RNA, Proteins poly- carbohydrate chains, lipids, PE, PP, PET and/or identify unmodified, pre/ or post modified molecules like peptides, drugs, hormones, antibodies, RNA typs like r, t, m, ds-RNA, RNAi, miRNA laying on the graphene layer.

3. The apparatus defined in claim 1 wherein said use for identification and/or sequencing using a graphene layer whereby the graphene layer is replaced by M0S2 or WS2 or nanotube layer or layers.

4. The apparatus defined in clainn 1 wherein said use for a light beam source to determine the sequence, whereby using one or more light beams for cutting and chemical modification or biochemical modification of the analyzed polymer and/or the graphene layer itself, in which the light beam source can be an integrated part and/or a separated of the apparatus of claim 1.

5. The apparatus defined in claim 1 to 4 wherein said from a composition,

comprising of a detector for electrons for measurement of current, voltage or the resulting electric field for detection, a computer for mathematical analysis of mathematical analysis of data and controlling and timing, whereby the detector for electrons for measurement of current, voltage or the resulting electric field for detection, a computer for mathematical analysis of

mathematical analysis of data and controlling and timing can be an integrated and/or a separated part of the apparatus of claim 1.

6. The apparatus defined in claim 1 to 5, wherein said light beam source used to determine the sequence, orientation and/or the surface of natural and artificial polymers, whereby replaced by AFM, CLM instead of the light beam or beams or whereby supplemented by a secondary determination method of the monomer sequence of the analyzed polymer like UV, IR, e-beam, X-Ray, AFM.

7. The apparatus defined in claim 1 to 6, wherein said uses for identification

and/or sequencing, whereby used for determination of the composition of a solution containing more than one type of molecules and quantification of the molecules in a solution.

8. The apparatus defined in claim 1 , wherein said use for identification and/or sequencing whereby the system is used for parallel sequencing of polymers and identification of small molecules or in separated steps.

9. The apparatus defined in claim 1 to 8, wherein said use for identification and/or sequencing whereby the system is used with a liquid probe solution like blood, urine, sputum or other fluids from plants, mammalian, cells, germs, insects or used with air probe for detection, identification, determination of 2D/3D surface properties, location, orientation and determination of chemical and physical properties of single molecules and/or sequencing natural or artificial polymers a single step or in separated steps.

10. Method using the apparatus of claim 1 to 8, where the method uses the graphene layer to apply a solution containing target molecules on them as the first step. After a probe containing molecules is placed on the graphene layer unit, the graphene unit is connected to the electric measurement detector. Second one or more laser beams from the laser beam unit scans continuously or point by point the surface of the graphene layer. The scan can be done by a laser unit with one or more distinctive combined light beams. The electric measurement unit detects the electric signals and the changes of them.

Emitting or reflecting lights from molecules by passing them with the laser beam are collected by the light detector and analyzed, e.g. fluorescence emission of different type of DNA or RNA molecules other light waves can be used also. The computer interprets and combines the data and information so the system is able to calculate the location, orientation and identification information of a molecule and/or the sequencing information of natural polymers like DNA or RNA.